The long-term goal of research in the Miller lab is to identify and characterize unique enzyme regulatory mechanisms. In this proposal, we investigate two mechanisms that regulate the activity of human glucokinase (GK), a protein that plays a key role in glucose homeostatic maintenance. Dysfunction in GK regulation causes maturity onset diabetes of the young type 2 and hyperlipidemia. The objective of Aim 1 is to elucidate the molecular origins of kinetic cooperativity in monomeric GK. GK cooperativity is fundamentally different from textbook models; it requires neither multiple ligand binding sites nor protein oligomerization. Instead, GK cooperativity results from hysteresis - it displays a time-dependent change in activity in response to changing glucose concentration that is driven by slow conformational changes. In preliminary studies, we have developed a working model for the hysteretic transitions that facilitate GK cooperativity. In our model, unliganded GK undergoes millisecond timescale conformational exchange between a crystallographically observed ground state (E) and an invisible excited state (E*). We have shown that the rate constant for conformational exchange (kex) is comparable in magnitude to the rate constant for turnover (kcat). The equivalency in these rate constants means that glucose binding does not reach equilibrium during the catalytic cycle, which is required for hysteresis to produce monomeric cooperativity. In Aim 1, we will develop a comprehensive and complete understanding of GK cooperativity by providing a structural description of the E* state and by establishing the mechanism(s) by which cooperativity is abolished in disease variants. The objective of Aim 2 is to use a combination of unnatural amino acid mutagenesis, stopped-flow spectrometry, crystallography and computational simulations to elucidate the mechanistic basis for GKRP-mediated regulation of GK. GKRP acts as a competitive inhibitor of glucose binding to GK. GKRP dysfunction is associated with cardiovascular disease and it has recently emerged as a viable diabetes therapeutic target. In Aim 2, we seek to understand how the association of different phosphorylated sugars with GKRP modulates affinity toward GK. We will also elucidate the kinetic mechanism of action of newly described drugs that disrupt the GK-GKRP complex. This proposal is significant to human disease because understanding the structural and dynamic basis of GK cooperativity and GKRP-mediated regulation is essential for the development of therapeutic agents that target both proteins. This work is significant to fundamental biochemistry because the structural and dynamic origins of hysteresis are unknown, despite the fact that regulation by hysteretic behavior is well established in enzymology. This proposal is innovative because it involves a unique combination of biophysical methods cutting-edge labeling methods to elucidate the mechanistic origins of kinetic cooperativity in a monomeric, single-site enzyme and to establish the mechanism of GK regulation by a protein-protein interaction.